During the current period of support, we used microarrays to define changes in gene expression in brain (prefrontal cortex and amygdala) and blood of alcoholic and non-alcoholic subjects. We have also profiled the expression of miRNAs in brain and propose that they are 'master switches,' responsible for many of the gene expression changes. In the proposed research, we will expand these findings in three areas. First, based on our preliminary studies of the GABAB receptor, we propose that regulation of some genes by chronic alcohol consumption is splice variant specific. Second, we will determine if the extent of changes in gene expression seen in human alcoholics are also present in three widely used mouse models of alcohol consumption. Because these mouse models are used for medication development, it is essential to determine the genetic factors that underlie neurochemical responses characteristic of alcoholism in these models. Third, selected miRNAs that were changed in frontal cortex of human alcoholics will be delivered to mouse brain to determine which gene networks are altered by these miRNAs. We propose that a single miRNA can produce selective changes in gene expression that change alcohol consumption. Three Specific Aims are proposed: 1) next generation sequencing will reveal novel splice variants of differentially expressed transcripts in human brain, and subsequent RT-PCR analysis will define splice-specific regulation of gene expression in alcoholism, 2) some expression changes in functional groups of genes found in the amygdala and frontal cortex of human alcoholics will be seen in these same brain regions using mouse models of excessive alcohol consumption, and 3) overexpression of select miRNAs in mouse brain will alter drinking phenotypes, and these changes will be correlated with specific patterns of gene expression in the prefrontal cortex and amygdala. PUBLIC HEALTH RELEVANCE: We propose that alcohol-induced changes in brain function are due to alterations in gene expression and we will explore these changes with several innovative approaches to alcohol research, including next generation sequencing of selected brain transcripts and delivery of alcohol-related miRNAs to brain. This work will provide new opportunities for gene-based diagnosis and treatment of alcohol dependence.